Apoptosis and matrix metalloproteinases (MMPs) have been implicated in many pathological diseases, such as cancer. Apoptosis or programmed cell death is a system which removes unnecessary, aged, or damaged cells. Apoptosis occurs during development to ensure proper formation of the fingers and toes in the fetus and of synapses between neurons in the brain; and serves to eradicate virus-infected cells, unnecessary immune cells, cells with DNA damage, and cancer cells. Decreased apoptosis has been implicated in developmental malformation, cancer and autoimmune disease, while enhanced apoptosis has been associated with degenerative diseases such as Alzheimer's disease, AIDS dementia, and Huntington's disease.
Caspases are a family of proteases, including initiator (activator) and effector (executioner) protease types, which regulate proteolysis during apoptosis. Apoptosis is triggered by signals which are either internal or external to the cell. In a healthy cell, the protein Bcl-2 is expressed on the surface and is bound to the protein Apaf-1. Internal damage in the cell causes Bcl-2 to release Apaf-1 and to no longer keep cytochrome c from leaking out of the mitochondria. The released cytochrome c and Apaf-1 bind to molecules of caspase 9. The resulting complex of cytochrome c, Apaf-1, caspase 9, and ATP aggregates in the cytosol. In cleaving a protein, caspase 9 activates other caspases, leading to digestion of structural proteins on the cytoplasm, degradation of chromosomal DNA, and phagocytosis of the cell. With regard to external signals (e.g., as in cytotoxic T cells inducing apoptosis in a virus-infected cell), binding of a death activator (FasL and Tumor necrosis factor or TNF) to the Fas and TNF receptor proteins on the surface of a target cell activates caspase 8, which activates other caspases leading to phagocytosis of the target cell.
Cancer cells may have mechanisms to avoid apoptosis. For example, some B-cell leukemias and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. Melanoma cells avoid apoptosis by inhibiting the expression of the gene which encodes Apaf-1. Lung and colon cancer cells secrete a molecule which binds to FasL, inhibiting its binding to Fas. Currently, radiation and standard chemotherapeutic drugs are used to induce apoptosis in some types of cancer cells; however, with such treatments having undesirable side effects and some cancers being resistant to such therapies, there exists a need to provide an effective approach which lacks such side effects, and demonstrates minimal interference with normal cell function.
Cancer tissue may also be treated with inhibitors of MMPs. Excessive MMPs have been implicated with diseases associated with the excessive degradation of extracellular matrix, such as tumor invasion and metastasis, arthritic diseases (rheumatoid arthritis and osteoarthritis), bone resorptive diseases (such as osteoporosis), enhanced collagen destruction associated with diabetes, periodontal disease, corneal ulceration, and ulceration of the skin.
MMPs are a family of at least 20 enzymes (proteases), including collagenases, gelatinases, stromelysins and stromelysin-like proteases as follows:    i) Collagenases include MMP-1 (interstitial), MMP-8 (neutrophil), and MMP-13, which catalyze the initial degradation of native collagen types I, II, III and VII. Collagen is an essential component of the extracellular matrix of tissues such as cartilage, bone, tendon and skin. MMP-13 is associated with osteoarthritis, ulcers and malignant tumor invasion.    ii) Gelatinases include MMP-2 (secreted by fibroblasts and a wide variety of other cell types) and MMP-9 (released by mononuclear phagocytes, neutrophils, corneal epithelial cells, tumor cells, cytotrophoblasts and keratinocytes). The gelatinases degrade gelatins (denatured collagens) and collagen type IV (basement membrane).    iii) Stromelysins include MMP-3, MMP-10 and MMP-11. MMP-3 and MMP-10 are expressed by epithelial cells and carcinomas, and degrade a broad range of extracellular matrix substrates, including laminin, fibronectin, proteoglycans, and collagen types IV and IX. MMP-11 is expressed by fibroblasts, and cleaves serine protease inhibitors.    iv) Stromelysin-like MMPs include MMP-12 and MMP-7. MMP-12 is expressed by macrophages and stromal cells, and degrades elastin. MMP-7 or matrilysin is expressed by mononuclear phagocytes and sporadically in tumors, and degrades a wide range of matrix substrates including proteoglycans, gelatins, fibronectin, elastin, and laminin.
MMPs are involved in the degradation of connective tissues, such as collagen, elastins, fibronectin, laminin, and other components of the extracellular matrix. Such components are present in the linings of joints, interstitial connective tissues, basement membranes, and cartilage. MMPs are present in various cell types which reside in or are associated with connective tissue, such as fibroblasts, monocytes, macrophages, endothelial cells, and invasive or metastatic tumor cells. Expression of MMPs may be induced by a variety of factors, including growth factors, chemical agents, physical stress, cell-matrix interactions, cell-cell interactions, oncogenic transformation, and cytokines. Cytokines affect the magnitude of inflammatory or immune responses, and can be divided into several groups, which include interferons, tumor necrosis factor (TNF), interleukins (IL-1 to IL-8), transforming growth factors, and the hematopoietic colony-stimulating factors.
MMPs and cytokines are associated and interact in various ways. Macrophages produce MMPs and also amplify inflammation by secreting cytokines, such as TNF-α and IL-1β. TNF-α promotes maturation of macrophages and neutrophils, while IL-1β promotes T and B cell proliferation and activation, and proteolysis. Cytokines, such as TNF-α and IL-1β, can regulate the transcription of MMPs, or lead to increased processing of MMPs from inactive to active forms. MMPs can also affect cytokines by inducing the release of membrane-bound cytokines, resulting in inhibition or activation of the cytokine depending on the particular type; using cytokines as substrates for MMP activity; and cleaving cytokines, such as TNF, from inactive to active forms. Inhibitors of MMPs may serve as potential therapeutic agents. A variety of naturally occurring or synthetic MMP inhibitors have been developed, including secondary amines (EP 159,396 to Searle); hydroxamic acid derivatives (EP 498,665 to Beckett et al.); collagenase inhibitors (EP 497,192 to Lobb et al.; U.S. Pat. No. 4,918,105 to Cartwright et al.); synthetic inhibitors (U.S. Pat. No. 5,773,438 to Levy et al.); tetracycline compounds (U.S. patent application Publication No. 2002/0045603 A1 to Golub et al.) and others known in the art. However, certain hydroxamic acids and derivatives thereof which have been suggested as collagenase inhibitors appear to be potentially toxic due to the hydroxamic moeity. Undesirable side effects specifically associated with use of MMP inhibitors have been reported, such as joint pain and exacerbation of liver injury (U.S. patent application Publication No. 2002/0035065 A1 to Bird et al.).
Modulation rather than total inhibition of MMPs and cytokines is desired for some conditions such as wound healing, where MMPs are required for angiogenesis and cell migration. Prolonged inflammatory response in a wound can delay healing, resulting in the destruction of tissue by processes which normally promote healing and synthesis of new tissue. MMPs are normally present in wounds for the purpose of breaking down damaged tissue in a controlled manner. However, elevated MMP activity impairs healing by degrading new tissue and growth factors, thereby damaging viable cells and the wound surface. There thus exists a need for a non-toxic, effective treatment to inhibit or modulate MMPs depending upon the disease of interest.
A treatment which provides a two-fold approach, namely induction of apoptosis of particular cells and inhibition of MMPs to eradicate cancer cells, and to reduce tissue damage contributing to tumor invasion and metastasis, may be desirable. Currently, cis-platin and its variations have been used as pro-apoptotic, anti-cancer agents, since the platinum complex attacks the DNA of tumor cells, thus disrupting RNA synthesis. However, cis-platin can pass through the blood to the kidneys and be immediately excreted; bind to proteins and be rendered inactive before reaching the tumor cells; or attack cells which are not tumors. Since acute toxicity may occur with long term use, cis-platin is restricted to short term, high doses. Sustained release to maintain treatment efficacy and non-toxicity in an anti-tumor agent are desirable.
Further, there are diseases and conditions for which MMP activity requires modulation, rather than total inhibition, to restore normal MMP activity. Although onset of inflammation is required for wound healing for example, excessive release, hence activity, of inflammatory mediators such as MMPs and cytokines contribute to wound damage, thus delaying healing.
While the patent literature reports that silver metal or silver salts such as silver nitrate, silver halides or silver sulphadiazine are among useful antibacterial agents, they have not, to the inventors' knowledge, been known or adopted to induce apoptosis and/or inhibit or modulate MMPs. For tumor tissue and cancerous lesions, there may be benefits associated with enhanced cellular apoptosis and inhibition of MMPs; for example, induction of apoptosis may aid in tumor suppression by eradicating tumor cells and by reducing the chance of tumor invasion and metastasis through inhibition of MMPs. In addition to affecting cancer cells, such treatment may be beneficial in eradicating excessive, normal cells, as in hyperplastic tissue in which abnormal multiplication or increase in the number of cells in a normal arrangement in normal tissue or an organ has occurred. In other diseases or a wound for example, modulation of MMPs rather than total inhibition may be desired.